We have found that a transcriptional repressor first defined functionally in our laboratory, NKAP, is absolutely required for the maintenance of the hematopoietic stem cell (HSC) pool. Steady state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of HSCs. Disruption of this balance can lead to hematopoietic failure, as in the case of hematopoietic differentiation without self-renewal which leads to the loss of the HSC pool. An understanding of the molecular regulation of HSC maintenance could lead to therapeutic interventions to hasten HSC reconstitution, for example, in bone marrow transplants. In adult mice, inducible deletion of NKAP results in hematopoietic failure and rapid lethality. NKAP deletion results in a complete, cell-intrinsic loss of all HSCs, through a combination of decreased survival and decreased proliferation. Therefore, NKAP is critical for HSC maintenance and survival. To understand the molecular basis for the requirement of NKAP in HSCs, we performed a yeast two hybrid screen, and identified that NKAP associates with the polycomb repressive complex-1 (PRC1) E3 ubiquitin ligase Ring1b. PRC1 is recruited to sites of H3K27 trimethylation, an inhibitory epigenetic mark, leading to monoubiquitination of H2AK119. Mutations in three components of PRC1, Ring1b, Bmi-1, and Rae28, disrupt HSC function, indicating the importance of this pathway for maintaining the HSC pool. In ex vivo short term HSC cultures, loss of NKAP leads to a global and severe downregulation of H2AK119 monoubiquitation, indicating that NKAP is a critical regulator of PRC1 in HSCs. This proposal will focus on elucidating the mechanism of Ring1b/PRC1 regulation by NKAP, and to uncover the molecular basis for the contribution of NKAP to HSC maintenance and survival. Specific Aim 1: Defining the mechanism of regulation of Ring1b/PRC1 function by NKAP Specific Aim 2: Identification of genes downstream of NKAP that are required for HSC maintenance and survival Specific Aim 3: Defining the critical pathways regulated by NKAP in HSCs through identification of the protein-protein domains and associations required for NKAP-mediated regulation of HSC maintenance and survival.